Synthesis and structure-activity relationships of 1-arylmethyl-3-(2-aminopropyl)-5-aryl-6-methyluracils as potent GnRH receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(03)00620-6

The novel synthesis and SAR studies of 6-methyluracils as human GnRH receptor antagonists are discussed. Introduction of a small methyl substituent at the β-position from N3 of the uracil improved the GnRH binding potency by 5- to 10-fold. The best compou

Full text of DOI:10.1016/S0960-894X(03)00620-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3311–3315
Synthesis and Structure–Activity Relationships of 1-Arylmethyl-3-
(2-aminopropyl)-5-aryl-6-methyluracils as Potent GnRH
Receptor Antagonists
Zhiqiang Guo,a,* Yun-Fei Zhu,a Fabio C. Tucci,a Yinghong Gao,a R. Scott Struthers,b
John Saunders,a Timothy D. Gross,a Qiu Xie,b Greg J. Reinhartb and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
bDepartment of Exploratory Discovery, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
Received 4 March 2003; accepted 19 May 2003
Abstract—The novel synthesis and SAR studies of 6-methyluracils as human GnRH receptor antagonists are discussed. Introduc-
tion of a small methyl substituent at the b-position from N3 of the uracil improved the GnRH binding potency by 5- to 10-fold. The
best compound from the series had binding affinity of 5 nM (Ki) to the human GnRH receptor.
# 2003 Elsevier Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH), also known
as luteinizing hormone-releasing hormone (LHRH), is
the decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-
Pro-Gly-NH2), which is produced in and secreted by the
hypothalamus in a pulsatile manner.1,2 It acts on the
pituitary gland to stimulate the secretion of both
luteinizing hormone (LH) and follicle-stimulating hor-
mone (FSH). These gonadotropines, in turn, act on the
reproductive organs, where they participate in the reg-
ulation of gonadal steroid production, spermatogenesis
in males and follicular development in females. Several
reproductive disease conditions such as endometriosis,
uterine fibroids and prostate cancer can be treated by
suppression of the pituitary–gonadal axis. Currently,
depot forms of peptidic GnRH agonists, represented by
leuprorelin1 are used to treat such conditions through a
receptor down-regulation mechanism to suppress gona-
dal steroid production.3 However, recent clinical evi-
dence has shown that peptidic GnRH antagonists can
act immediately at the receptor to lower steroid levels
and therefore alleviate disease symptoms without the
concomitant ‘flare effect’, which is exhibited by the
peptide agonists due to their initial over-stimulation of
the receptor.4 Nevertheless, both peptide agonists and
antagonists require parenteral administration, typically
in depot form due to their poor oral bioavailability.
Small molecule GnRH antagonists offer the potential
for oral administration and therefore could gain wider
acceptance from patients. In response to that need,
intensive efforts have been initiated for the development
of small molecule GnRH antagonists.5,6
In earlier papers,7,8 we disclosed the SAR study of
7-phenylpyrrolo[1,2-a]pyrimid-4-ones I and II7 (Fig. 1),
and 2-phenylimidazolo[1,2-a]pyrimidines III and IV8 as
potent hGnRH receptor antagonists. These studies
showed that the pyridyl-ethyl group in the basic side
chain is very important for high receptor-binding activ-
ity and 3-methoxyphenyl is a good substitution on the
right side of the molecules. Most recently,9 we have
discussed the initial SAR studies of a novel series of
monocyclic uracils (1 and 2) as human GnRH receptor
antagonists. The best compound (2a) from the initial
SAR study has a Ki value of 34 nM, which is less potent
than some of our bicyclic analogues. To enhance the
potency, we rationalized that restriction of the flexible
side chain from N3 may be beneficial, and furthermore,
a small alkyl group such as a methyl at the b-position
could serve such a purpose. In this letter, we report a
novel and improved synthesis of the 6-methyl uracil
core structure to give the 1-arylmethyl-3-(2-aminopro-
pyl)-5-aryl-6-methyluracil structure. SAR of this series
of uracils with an amino-isopropyl side chain at the N3
position is described.
*Corresponding author. Tel.: + 1-858-658-7657; fax: + 1-858-658-
7601; e-mail: zguo@neurocrine.com. http://www.neurocrine.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00620-6

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Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3311–3315
Figure 1. Small molecule GnRH antagonists.
The 6-methyluracil core structure was initially synthe-
sized by condensation of allylurea and ethyl aceto-
acetate.9,10 The yield was low and the substitution at N3
was limited by the availability of substituted ureas. To
perform a more comprehensive SAR study at N3, a new
synthetic route had to be developed. Thus, 2,6-difluor-
obenzyl amine and urea were refluxed in HCl/water
solution to form the benzyl urea 4 (Scheme 1), and then
crystallized from EtOAc in 79% yield. Urea 4 and
diketene were then refluxed in acetic acid for 40 min to
yield a mixture of N1- and N3-substituted 6-methylur-
acils (5 and 6) in 3:1 ratio. The structures of 5 and 6
were confirmed by NOE NMR experiments. In com-
pound 5, for instance, there was a clear NOE of the 6-
methyl signal upon irradiation of the benzylic protons
at N1; this effect was absent for 6. 1-(2,6-difluoro-
benzyl)-6-methyluracil (5) was separated from the
N3-isomer (6) by recrystallization in acetic acid. This
two-step sequence afforded 5 in about 25% yield from
3. After further experimentation, a more efficient route
to uracil 5 was developed to afford the N1-substitued
6-methyluracil exclusively. Thus, compound 4 was first
treated with diketene in pyridine at ambient tempera-
ture for 24 h, and the intermediate 7 was separated in
60% yield. Compound 7 was then refluxed in acetic acid
for 1 h and the desired product 5 crystallized out from
the reaction mixture in 95% yield (Scheme 1).
Scheme 1. Reagents and conditions: (a) urea, water, HCl, reflux, 79%;
(b) diketene, HOAc, reflux, 40 min, 69%; (c) diketene, pyridine, rt, 24
h, 60%; (d) HOAc, reflux, 1 h, 95%.
its corresponding S-isomer under Mitsunobu conditions
in THF using di-t-butyl-azodicarboxylate as the coupl-
ing reagent to give 13. Suzuki coupling of 13 with
3-methoxyphenylboronic acid under the catalysis of
Pd(0) yielded 14. Deprotection of the amino-Boc group
with TFA followed by two successive reductive amina-
tions, first with aldehydes to give 16 and then with
formaldehyde generated the N-methylated final pro-
ducts 17. Finally, cyclic analogues that link the
N-methyl and the b-methyl group with a methylene
moiety were prepared from 8 with either N-t-Boc-R-
prolinol or its corresponding S-isomer to give 18
(Scheme 4). Suzuki coupling of 18 yielded 19, deprotec-
tion of which afforded 20 and reductive amination of 20
with 2-pyridinecarboxaldehyde yielded the desired final
products 21.
Uracil 5 was brominated in acetic acid to yield 8
(Scheme 2), which was alkylated at the N3 position by
treatment with 3-bromo-2-methylpropene and K2CO3
in DMF at ambient temperature. The resulting alkene
(9) was then oxidized to the methyl ketone with NaIO4/
OsO4 in 1:1 THF/water, followed by Suzuki coupling
with 3-methoxyphenylboronic acid yielded the key
intermediate 10. The ketone (10) was treated under
reductive amination conditions with a variety of amines
(R1R2NH) in dichloroethane, followed by treatment
with NaBH(OAc)3 to afford the desired amines 11.
N-Methylation was achieved by reductive amination of
11 with formaldehyde to afford final products 12.
All synthesized compounds were evaluated for their
ability to inhibit des-Gly10[125I-Tyr5,DLeu6,NMeLeu7,
Pro9-NEt]-GnRH radioligand binding to the cloned
hGnRH receptor stably expressed in HEK293 cells
using a 96-well filtration assay format.11
To prepare enantiomerically pure molecules, an alter-
native synthetic approach was used (Scheme 3). Com-
pound 8 was treated with either N-t-Boc-R-alaninol or
The binding affinities of 1–2 and 11–12 for the hGnRH
receptor are summarized in Table 1. As previously
observed with 1 and 2, N-methylated compounds were

Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3311–3315
3313
Scheme 2. Reagents and conditions: (a) Br2, HOAc; (b) 3-bromo-2-methylpropene, K2CO3, DMF; (c) NaIO4, OsO4 (cat), THF/H2O; (d) 3-methoxy-
phenyl boronic acid, Pd(Ph3P)4, Na2CO3, toluene/H2O, reflux; (e) R1NH2, NaBH(OAc)3, dichloroethane; (f) CH2O, NaBH(OAc)3, dichloroethane.
Scheme 3. Reagents and conditions: (a) N-t-Boc-R-alaninol or N-t-Boc-S-alaninol, PPh3, di-t-butyl-azodicarboxylate, THF; (b) 3-methoxyphenyl
boronic acid, Pd(Ph3P)4, Na2CO3, toluene/H2O, reflux; (c) TFA/CH2Cl2 (1:1); (d) aldehyde, NaBH(OAc)3, dichloroethane; (e) CH2O,
NaBH (OAc)3, dichloroethane.
in general more potent than their NH analogues. Thus
compound 12a was 14 times; 12b was 3 times more
potent than 11a and 11b, respectively. Importantly, the
potency of the earlier compounds (1–2) was greatly
improved by the incorporation of a b-methyl group to
the ethylene group. This trend was very clear, especially
when there was a pyridine ring on the N3-branch sub-
stitution. 11a was 6 times more potent than 1a and 11b
was 10 times more potent than 1b. Enhancement of bind-
ing affinity by the b-methyl group was also obvious in the
N-methyl series compare 2a and 2b with 12a and 12b,
respectively. 12a and 12b had comparable potency; the
extra methylene did not make noticeable difference for
activity. With the b-methyl group, N-alkyl substituents
such as isobutoxypropyl (11d) also demonstrated good
binding activity, which was even better than that of the
benzyl analogue (11c).
The hGnRH receptor binding data for the enantiomeric
compounds 15–17 and 21 are reported in Table 2. With
the 2-pyridylmethyl- or 2-pyridylethyl-substituent, the
R-isomers were obviously more active than the S-iso-
mers by at least one order of magnitude. Interestingly,
N-methylation improved GnRH binding specifically for
the R-isomer, thereby enhancing the potency ratio to
about 100-fold. Surprisingly, for the N-benzyl com-
pounds, there was almost no difference between the R
and S isomers in binding activity and N-methylation of

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Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3311–3315
Scheme 4. Reagents and conditions: (a) N-t-Boc-R-prolinol or N-t-Boc-S-prolinol, PPh3, di-t-butyl-azodicarboxylate, THF; (b) 3-methoxyphenyl
boronic acid, Pd(Ph3P)4, Na2CO3, toluene/H2O, reflux; (c) TFA/CH2Cl2 (1:1); (d) 2-pyridinecarboxaldehyde, NaBH(OAc)3, dichloroethane.
Table 1. Binding affinities of 1-arylmethyl-3-(2-aminoethyl)-5-aryl-6-
methyluracils (1–2) and 1-arylmethyl-3-(2-aminopropyl)-5-aryl-6-
methyluracils (11–12) on the human GnRH receptor12
Compd
R1
R2
Ki (nM)
1a
1b
2a
2b
11a
11b
11c
11d
12a
12b
12c
12d
H–
H–
CH3–
CH3–
H–
H–
H–
H–
CH3–
CH3–
CH3–
CH3–
(2-Pyr)–CH2CH2–
(2-Pyr)–CH2–
(2-Pyr)–CH2CH2–
(2-Pyr)–CH2–
(2-Pyr)–CH2CH2–
(2-Pyr)–CH2–
Ph-CH2–
1100
550
34
96
170
53
280
81
12
18
310
240
i-Butyl-O–CH2CH2CH2–
(2-Pyr)-CH2CH2–
(2-Pyr)-CH2–
Ph-CH2–
i-Butyl-O–CH2CH2CH2–
Figure 2. Inhibition of GnRH stimulated Ca2+ flux by compound
(R)-17b.
Table 2. Binding affinities of 1-arylmethyl-3-(2-aminopropyl)-5-aryl-
6-methyluracils (15–17) and 1-arylmethyl-3-[N-(2-pyridylmethyl)-2-
pyrrolidinylmethyl]-5-aryl-6-methyluracils (21) on the human GnRH
receptor12
selected for testing its ability to inhibit Ca2+ influx
induced by GnRH.13 The result shown in Figure 2
indicates that (R)-17b is a potent functional antagonist
with IC50=2.5 nM. Similar to our previous results,7a
these compounds demonstrated species differences in
their binding to the GnRH receptors. Compound
(R)-17b exhibited reduced binding affinity on the mon-
key GnRH receptor (Ki=102 nM) and much lower
affinity on the rat GnRH receptor (Ki=21 mM).
Compd
R1
R2
Ki (nM) chirality
R
S
15
H–
H–
H–
H–
CH3–
CH3–
CH3–
H–
2500
79
15
130
5.2
5.5
330
32
18,000
780
460
16a
16b
16c
17a
17b
17c
21
(2-Pyr)–CH2CH2–
(2-Pyr)–CH2–
Ph–CH2–
(2-Pyr)–CH2CH2–
(2-Pyr)–CH2–
Ph–CH2–
140
890
470
250
In conclusion, we have discovered a novel, highly con-
vergent cyclization procedure for assembly of the
6-methyl uracil core. Introduction of a small methyl
substituent at the b-position from N3 of the uracil
improved the GnRH binding potency by 5- to 10-fold.
(R)-Enantiomers were much more potent than their
(S)-counterparts when a 2-pyridyl group was attached.
This phenomenon may be explained by a receptor
model where the (R)-methyl group orients the pyridyl
side chain in close proximity to the aspartic acid 302 on
helix 7, which we believe is a critical recognition point
on the receptor, thus allowing for optimal binding.
210
these compounds [(R)- and (S)-17c] did not improve
their potency. Finally, the proline-based compound
(R)-21 was less potent than (R)-17(a–b), and the (R)-21
was 6 times more potent than its S-isomer.
To ensure that these high-binding affinity molecules
were functional antagonists, compound (R)-17b was

Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3311–3315
3315
Further structure–activity relationships of this series of
compounds as GnRH antagonists will be presented
elsewhere.
Reinhart, G. J.; Chen, C. Bioorg. Med. Chem. Lett. 2002, 12,
403.
8. (a) Wilcoxen, K.; Zhu, Y.-F.; Connors, P. J., Jr.; Saunders,
J.; Gross, T. D.; Gao, Y.; Reinhart, G. J.; Struthers, R. S.;
Chen, C. Bioorg. Med. Chem. Lett. 2002, 12, 2179. (b) Gross,
T. D.; Zhu, Y.-F.; Saunders, J.; Gao, Y.; Connors, P. J., Jr.;
Guo, Z.; Struthers, R. S.; Reinhart, G. J.; Chen, C. Bioorg.
Med. Chem. Lett. 2002, 12, 2185. (c) Zhu, Y.-F.; Guo, Z.;
Gross, T. D.; Gao, Y.; Connors, P. J., Jr.; Struthers, R. S.;
Xie, Q.; Tucci, F. C.; Reinhart, G. J.; Wu, D.; Saunders, J.;
Chen, C. J. Med. Chem. 2003, 46, 1769.
References and Notes
1. Huirne, J. A. F.; Lambalk, C. B. Lancet 2001, 358, 1793.
2. (a) Matsuo, H.; Baba, Y.; Nair, R. M. G.; Arimura, A.;
Schally, A. V. Biochem. Biophys. Res. Commun. 1971, 43,
1334. (b) Amoss, M.; Burgus, R.; Blackwell, R.; Vale, W.;
Fellows, R.; Guillemin, R. Biochem. Biophys. Res. Commun.
1971, 44, 205.
9. Zhu, Y.-F.; Gross, T.; Guo, Z.; Gao, Y.; Connors, P. J.,
Jr.; Tucci, F. C.; Struthers, R. S.; Reinhart, G. J.; Saunders, J.;
Chen, C. J. Med. Chem. 2003, 46, 2023.
3. Fujino, M.; Fukuda, T.; Shinagawa, S.; Kobayashi, S.;
Yamazaki, I.; Nakayama, R.; Seely, J. H.; White, W. F.; Rip-
pel, R. H. Biochem. Biophys. Res. Commun. 1974, 60, 406.
4. Goulet, M. T. Gonadotropin Releasing Hormone Antago-
nists. In Annual Reports in Medicinal Chemistry, Bristol, J. A.,
Ed.; Academic: New York, 1995; p 169.
5. Cho, N.; Harada, M.; Toshihiro, I.; Imada, T.; Matsu-
moto, H.; Hayase, Y.; Sasaki, S.; Furuya, S.; Suzuki, N.;
Okubo, S.; Ogi, K.; Endo, S.; Onda, H.; Fujino, M. J. Med.
Chem. 1998, 41, 4190.
10. Grahner, B.; Winiwarter, S.; Lanzner, W.; Mueller, C. E.
J. Med. Chem. 1994, 37, 1526.
11. Perrin, M. H.; Haas, Y.; Rivier, J. E.; Vale, W. W. Mol.
Pharmacol. 1983, 23, 44.
12. On each assay plate a standard antagonist of comparable
affinity to those being tested was included as a control for
plate-to-plate variability. Overall, Ki values were highly
reproducible with an average standard deviation of 45% for
replicate Ki determinations. Key compounds were assayed in
3–8 independent experiments.
6. (a) Ashton, W. T.; Sisco, R. M.; Kieczykowski, G. R.;
Yang, Y. T.; Yudkovitz, J. B.; Cui, J.; Mount, G. R.; Ren,
R. N.; Wu, T. J.; Shen, X.; Lyons, K. A.; Mao, A. H.; Carlin,
J. R.; Karanam, B. V.; Vincent, S. H.; Cheng, K.; Goulet,
M. T. Bioorg. Med. Chem. Lett. 2001, 11, 2597. (b) Ashton,
W. T.; Sisco, R. M.; Yang, Y. T.; Lo, J. L.; Yudkovitz, J. B.;
Cheng, K.; Goulet, M. T. Bioorg. Med. Chem. Lett. 2001, 11,
1723. (c) Ashton, W. T.; Sisco, R. M.; Yang, Y. T.; Lo, J. L.;
Yudkovitz, J. B.; Gibbons, P. H.; Mount, G. R.; Ren, R. N.;
Butler, B. S.; Cheng, K.; Goulet, M. T. Bioorg. Med. Chem.
Lett. 2001, 11, 1727. (d) Chu, L.; Hutchins, J. E.; Weber, A. E.;
Lo, J. L.; Yang, Y. T.; Cheng, K.; Smith, R. G.; Fisher, M. H.;
Wyvratt, M. J.; Goulet, M. T. Bioorg. Med. Chem. Lett. 2001,
11, 509. (e) Chu, L.; Lo, J. L.; Yang, Y. T.; Cheng, K.; Smith,
R. G.; Fisher, M. H.; Wyvratt, M. J.; Goulet, M. T. Bioorg.
Med. Chem. Lett. 2001, 11, 515.
13. Inhibition of GnRH stimulated Ca2+ flux: Functional
activity of compounds for the human GnRH receptor was
determined by inhibition of GnRH stimulated Ca2+ flux. RBL
cells stably expressing the full-length human GnRH receptor
were seeded into 96-well, black-wall clear-bottom plates
(Corning) at a density of 50,000 cells/well and the cells allowed
to attach overnight. Cells were then loaded with the Ca2+ sensi-
tive dye, Fluo-4 (Molecular Probes), by incubation in loading
medium [(DMEM with 20 mM Hepes, 10% FBS, 2 mM Fluo-4,
0.02% pluronic acid (Molecular Probes) and 2.5 mM probenecid
(Sigma)] for 1 h at 37ꢀC. Cells were then washed three times with
assay buffer (Hanks balanced salt, 20 mM Hepes, 2.5 mM pro-
benecid). Compounds at varying concentrations in assay buffer
were pre-incubated with cells for 1 min prior to stimulation with
GnRH (5 nM). Measurement of fluorescence due to GnRH sti-
mulated Ca2+ flux was performed according to manufacturer’s
instructions on a FLIPR system (Molecular Devices, FLIPR384
system). IC50 values for the inhibition of GnRH-stimulated
Ca2+ flux were calculated using the Prism software package
(GraphPad Software) with a ‘sigmoidal dose–response (variable
slope)’ option for curve fitting.
7. (a) Zhu, Y.-F.; Struthers, R. S.; Connors, P. J., Jr.; Gao,
Y.; Gross, T. D.; Saunders, J.; Wilcoxen, K.; Reinhart, G. J.;
Ling, N.; Chen, C. Bioorg. Med. Chem. Lett. 2002, 12, 399. (b)
Zhu, Y.-F.; Wilcoxen, K.; Saunders, J.; Guo, Z.; Gao, Y.;
Connors, P. J., Jr.; Gross, T. D.; Tucci, F. C.; Struthers, R. S.;